Separate mesolimbic dopaminergic pathways mediate the opposing motivational effects of acute caffeine by Mandy Yee A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Mandy Yee 2018 Separate mesolimbic dopaminergic pathways mediate the opposing motivational effects of acute caffeine Mandy Yee Master of Science Institute of Medical Science University of Toronto 2018 Abstract Caffeine is the most commonly consumed psychoactive drug in the world, yet little is known about the neural substrates that underlie its rewarding and aversive properties. Using male Wistar rats in a place conditioning procedure, we showed that systemic caffeine at a low intraperitoneal dose of 2 mg/kg (or 100µM injected directly into the rostral, but not caudal, ventral tegmental area) induced reward. By contrast, high doses of systemic caffeine (10 and 30 mg/kg) produced aversions that were not recapitulated by a caffeine analog restricted to the periphery. We demonstrated that pharmacological blockade of dopamine receptors using α-flupenthixol injected into the nucleus accumbens shell, but not core, blocked caffeine reward. Conversely, α- flupenthixol injected into the nucleus accumbens core, but not shell, blocked caffeine aversions. Thus, our findings reveal two dopamine-dependent and functionally dissociable mechanisms for processing caffeine motivation that are segregated between nucleus accumbens subregions. ii Acknowledgments I would like to acknowledge my friends and family for supporting me through all the ups and downs of research. First and foremost, I would like to thank my parents for their loving support. I would also like to thank my siblings – Lance, Angela, Holly, and Kingly – for their support. A great big thanks to my graduate supervisor and mentor, Dr. Derek van der Kooy, whose combination of excellent guidance, thoughtful critiques, and vast knowledge inspired me to grow and learn as a scientist. Thank you to my program advisory committee members, Drs. Cindi Morshead and Paul Fletcher, for their insights, advice, and patience. I would also like to thank my lab mentors, Taryn and Geith, who patiently taught me everything I needed to know for my project. A huge thanks to our lab staff extraordinaire, Brenda and Monica, for being incredibly kind and helpful. I also cannot stress enough the importance of my amazing fellow lab members that I have worked with over the years. I want to especially thank Isabel, Michael, and the other undergraduates in the motivation group for keeping me company in the necropsy and surgery room those long days and nights. Additionally, I would like to thank Justin, Daniel, Ken, Sylvia, and the other lab members for always being in the lab even at the increasingly odd hours that I came in to do lab work. Even though we were not working on the same projects, you guys are excellent company and made the evenings far more interesting. I would also like to thank the staff at the Division of Comparative Medicine for taking excellent care of the animals, and the Zandstra lab members who patiently helped me with my secondary project. A special thank you to Marco for putting up with all my complaining – and there was a lot of it! I am especially grateful for the time you spent giving me the push I needed to finish this thesis. I dedicate this thesis to my family and friends who have supported me every step of the way. iii Statement of Contributions Mandy Yee (author) solely prepared this thesis. All aspects of this body of work were performed by the author in whole or in part. The following contributions by other individuals is formally acknowledged below: Dr. Derek van der Kooy (primary supervisor) - Mentorship, including guidance in planning experiments and interpreting results - Provision of laboratory materials and equipment - Preparation of manuscript and thesis Dr. Cindi Morshead (program advisory committee member) - Mentorship, including guidance in planning experiments and interpreting results Dr. Paul Fletcher (program advisory committee member) - Mentorship, including guidance in planning experiments and interpreting results Dr. Taryn Grieder - Mentorship, including guidance in planning experiments and interpreting results Geith Maal-Bared - Mentorship, including guidance in planning experiments and interpreting results - Assistance in executing experiments Dr. Ryan Ting-A-Kee - Assistance in executing experiments and laying foundation of project Michal Chwalek - Assistance in executing experiments and laying foundation of project Isabel MacKay-Clackett - Assistance in executing experiments Michael Bergamini - Assistance in executing experiments iv Table of Contents Abstract .................................................................................................................................... ii Acknowledgments .................................................................................................................... iii Statement of Contributions ......................................................................................................... iv Table of Contents ....................................................................................................................... v List of Abbreviations............................................................................................................... viii List of Figures and Tables ......................................................................................................... xi Chapter 1 - Literature Review ................................................................................................. 1 1.1 Introduction to motivation ..................................................................................................... 2 1.2 Introduction to drug addiction .............................................................................................. 4 1.3 Measurements of motivation ................................................................................................. 7 Operant conditioning (self-administration)....................................................................... 8 Classical conditioning (place conditioning) .................................................................... 11 1.4 Models and theories of drug motivation ............................................................................... 16 The mesolimbic dopamine reward hypothesis ............................................................... 16 The error prediction model ............................................................................................ 19 The incentive-sensitization theory .................................................................................. 22 The non-deprived/deprived hypothesis........................................................................... 23 The opponent process theory ......................................................................................... 26 1.5 Neuroanatomy of motivation ............................................................................................... 32 Anatomy of the ventral tegmental area ........................................................................... 32 Anatomy of the nucleus accumbens .............................................................................. 38 v 1.6 Neurobiology of caffeine motivation .................................................................................. 41 Introduction to caffeine consumption ............................................................................. 41 1.7 Research hypotheses and objectives .................................................................................... 43 Chapter 2 - Materials and Methods ....................................................................................... 46 2.1 Animal subjects and drugs ................................................................................................... 47 2.2 Experimental procedures .................................................................................................... 49 Surgical cannulation ...................................................................................................... 49 Place conditioning ......................................................................................................... 49 2.3 Histology ............................................................................................................................ 51 Verification of cannula placements ............................................................................... 51 2.4 Statistical methods .............................................................................................................. 52 Chapter 3 - Results ................................................................................................................. 53 3.1 Systemic caffeine dose-response ......................................................................................... 54 Systemic caffeine elicits reward at low doses and aversion at higher doses ................... 54 3.2 Intra-rVTA caffeine reward ................................................................................................. 59 Caffeine injections into the rostral VTA elicit reward .................................................... 59 Intra-rVTA caffeine reward is mediated through the NAc shell, but not core ................. 65 3.3 High dose systemic caffeine aversion